Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium.

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Meningococcal type IV pili recruit the polarity complex

to cross the brain endothelium.

Coureuil Mathieu, Guillain Mikaty, Florence Miller, Hervé Lécuyer, Christine

Bernard, Sandrine Bourdoulous, Guillaume Duménil, René-Marc Mège,

Babette Weksler, Ignacio Romero, et al.

To cite this version:

Coureuil Mathieu, Guillain Mikaty, Florence Miller, Hervé Lécuyer, Christine Bernard, et al.. Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium.. Science, American Association for the Advancement of Science, 2009, 325 (5936), pp.83-7. �10.1126/sci-ence.1173196�. �inserm-00767121�

Meningococcal Type IV Pili Recruit the Polarity Complex to Open the Cell-Cell

Junctions of Brain Endothelium.

Mathieu Coureuil1*, Guillain Mikaty1, Florence Miller2,3, Hervé Lécuyer1,5, Christine

Bernard1, Sandrine Bourdoulous2,3, Guillaume Duménil1,8, René-Marc Mège4, Babette B.

Weksler6, Ignacio A. Romero7, Pierre Olivier Couraud2,3, Xavier Nassif1,5

1 Université Paris Descartes, Faculté de Médecine, INSERM (U-570), Paris, France.

2 Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.

3 INSERM, U567, Paris, France.

4 INSERM UMR-S 839, Université Pierre et Marie Curie-Paris6, Institut du Fer à

Moulin, Paris, France.

5 AP-HP, Hôpital Necker-Enfants Malades, Paris, F-75015, France.

6 Weill Cornell Medical College, New York, USA.

7 Department of Life Sciences, The Open University, Walton Hall, Milton Keynes, UK.

8 Present address: INSERM U970, Paris Cardiovascular Research Center, Paris, F-75015,

France

Type IV pili mediate the initial interaction of many bacterial pathogens with their

host cells. In Neisseria meningitidis, the causative agent of cerebrospinal meningitis,

type IV pili-mediated adhesion to brain endothelial cells is required for bacteria to

cross the blood-brain barrier. Here, Type IV pili-mediated adhesion of N.

meningitidis to human brain endothelial cells was found to recruit the

Par3/Par6/PKCζ polarity complex that plays a pivotal role in the establishment of

eukaryotic cell polarity and the formation of intercellular junctions. This

recruitment leads to the formation of ectopic intercellular junctional domains at the

site of bacterial-cell interaction and a subsequent depletion of junctional proteins at

the cell-cell interface with opening of the intercellular junctions of the

Neisseria meningitidis is a commensal bacterium of the human nasopharynx that, after

bloodstream invasion, crosses the blood-brain barrier (BBB) (1). Few pathogens have a

tropism for the brain, indicating that N. meningitidis possess specific components to

interact with the BBB. Meningeal colonization by invasive capsulated N. meningitidis is

the consequence of the bacterial adhesion onto brain endothelial cells (2, 3) which is

followed by bacterial division onto the apical surface of the cells (Movie S1). This

process is mediated by Type IV pili (Tfp) (4-9). In addition, by powering a form of cell

locomotion, reported as twitching motility (10), Tfp lead to the spread of the bacteria on

the surface of the cells and the formation of microcolonies. Subsequent to the formation

of these microcolonies, Tfp trigger the recruitment of cortical actin and signal transducing

proteins leading to the formation of filopodia-like structures (2, 11-13). The crossing of

the BBB by N. meningitidis implies that following Tfp mediated adhesion, the bacteria

transcytose through the brain capillaries and/or open the brain endothelium.

To investigate whether adhesion of N. meningitidis affects the integrity of the adherens

(AJ) and/or tight (TJ) junctions of human brain endothelial cells, the consequences of

infection by N. meningitidis on the distribution of junctional proteins were analyzed using

the human brain microvascular endothelial cell line hCMEC/D3 (14). After infection,

components of the AJ (VE-cadherin, p120-catenin, β-catenin) and TJ (ZO1, ZO2, and

claudin-5) were targeted underneath N. meningitidis colonies (Fig. 1A). At the site of N.

meningitidis adhesion, these junctional proteins co-distributed with each other and with

the actin honeycomb-like network. In non infected cells, the recruitment of junctional

proteins usually occurs at the cell-cell interface and is controlled by several polarity

underneath N. meningitidis colonies (Fig. 1B). Thus, N. meningitidis triggers a signal

leading to the formation of an ectopic domain containing filopodia-like structures and

enriched in junctional proteins, thus resembling spot-like adherens junctions observed

during early steps of junctional biogenesis. We refer to this domain as an “ectopic early

junction-like domain” (18). Using isogenic derivatives, Tfp-induced signaling was shown

to be responsible for the formation of these ectopic early junction-like domains (Fig. S1A

and B). However, Tfp retraction through the PilT motor was not required for formation of

the ectopic domains (Fig. S1D and E).

The small GTPase Cdc-42 is required for polarization of mammalian cells (19, 20). The

role of this component in the recruitment of the polarity complex by N.meningitidis was

investigated. Transfection of a dominant negative mutant of Cdc42 or knockdown of

Cdc42 by RNAi inhibited the recruitment of Par6, Par3 (Fig. 2A, S2A), VE-cadherin,

p120-catenin and actin (Fig. 2B, S2B, S3). These results link the Cdc42/polarity complex

pathway with the formation of the ectopic early junction-like domains.

The role of the polarity complex in the recruitment of junctional proteins was further

explored by studying the inhibition of Par3 and Par6 using either dominant negative

mutants or knockdown by RNAi. PKCζ inhibition was assessed using a PKCζ pseudosubstrate inhibitor (PKCζ-PS) (21). Inhibition of Par6 and PKCζ reduced the

recruitment of p120-catenin, VE-cadherin and actin (Fig. 2B, 2C, S2C, S3) and that of

Par3 (Fig. 2D, S2E), consistent with the finding that the Par6/PKCζ complex recruits

Par3 at intercellular junction domains (22). On the other hand, inhibition of Par3 reduced

only the recruitment of VE-cadherin (Fig. 2B, S2D, S3), consistent with Par3 being

These observations confirmed the role of the polarity complex in the recruitment of the

junctional proteins by N. meningitidis.

The sequence of events leading to the targeting of AJ proteins at the cell-cell junctions

during cellular polarization remains unknown. To get insight into this process, we

engineered a cadherin knockdown of hCMEC/D3 cells by stable expression of a

VE-cadherin shRNA (VEC shRNA) (Fig. 3A, 3B, S4A). In this cell line, p120-catenin and

actin were still recruited beneath N. meningitidis colonies, whereas recruitment of

β-catenin was dramatically reduced. On the other hand, down-regulation of p120-β-catenin

using RNAi (Fig. 3C, S4B) resulted in inhibition of VE-cadherin and of actin

recruitment. Consistent with a previous report, cortactin and Arp2/3 were not recruited by

the bacterial colonies in p120-catenin knockdown cells (24) (Fig. S4C). Furthermore,

inhibition of Src kinase, which phosphorylate cortactin and is activated following the

formation of the cortical plaque (25) did not modify p120-catenin recruitment but

inhibited VE-cadherin and actin recruitment (Fig. S4D, S4E). Taken together, these

results strongly suggest that p120-catenin-mediated recruitment of actin and VE-cadherin

requires the recruitment and phosphorylation of cortactin by the Src kinase. In summary,

Cdc42, via the polarity complex, organizes this ectopic early junction-like domain,

mainly by the initial recruitment of p120-catenin.

We asked whether the signal triggered by Tfp and leading to the formation of these

ectopic early junction-like domains destabilized intercellular junctions, especially by

redirecting a recycling pool of junctional proteins to the N. meningitidis adhesion site.

First, inhibition of protein synthesis did not prevent recruitment of VE-cadherin (Fig.

recruitment (Fig. S5B and S5C) suggesting that VE-cadherin internalization is required

for its targeting underneath N. meningitidis colonies. Third, when monolayers were

tagged before infection with a VE-cadherin monoclonal antibody, antibodies are

relocalized beneath colonies in infected monolayers (Fig. S6). Thus the VE-cadherin

delocalized by the bacteria was coming from the intercellular junctions. This

redistribution of the AJ proteins was associated with a reduction of the amount of tagged

cadherin at the intercellular junction (Fig. S6, Movie S2). Thus the junctional

VE-cadherin is internalized and then mistargeted at the site of bacterial cell interactions.

Depletion of intercellular junction proteins from the cell-cell interface could open a

paracellular route for bacterial spread. Indeed, N. meningitidis was shown to increase

permeability to Lucifer Yellow (LY) a compound which mark passive paracellular

diffusion (Fig. 4A) (26). Moreover, this increase relied on PKCζ activity and bacterial

piliation (Fig. 4A). This modification of permeability was associated with the formation

of gaps between infected cells (Fig. 4B). The number of gaps increased over time and was reduced by the PKCζ pseudosubstrate inhibitor (Fig. 4B and 4C). Gaps did not form

when cells were infected with a non piliated strain, showing that these gaps are due to

Tfp-mediated signaling (Fig. 4C). Indeed, piliated strain cross the monolayer at a higher

rate than non-piliated isogenic derivatives or a piliated strain in the presence of PKCζ PS

(Fig. 4D). Thus the signaling induced by N. meningitidis Tfp leading to the recruitment of

the polarity complex is associated with large alterations of the intercellular junctions

sufficient for the bacteria to cross the brain endothelial cell monolayer.

In summary, N. meningitidis microcolonies trigger via type IV pili a signal resembling

formation of ectopic early junction-like domains (Fig. S7), thus disorganizing the cell-cell

junctions and opening the paracellular route allowing N. meningitidis to cross the BBB

References and Notes

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27. Described in the Supporting Online Material: Materials and methods section.

28. The authors thank M. Drab, P. Martin, I. Allemand and N. Simpson for reviewing the

manuscript. The authors are grateful to M. Garfa-Traore and N. Goudin for technical

support. Mathieu Coureuil was funded by “la Fondation pour la Recherche Médicale”

FIGURE LEGENDS

Figure 1

Neisseria meningitidis recruits ectopic junction-like domains beneath colonies. (A)

VE-cadherin (green), the main component of the endothelial AJ, co-localized with actin

(red) beneath N. meningitidis colony (upper panel). Two other AJ components: p120-catenin and β-p120-catenin, and three components of the TJ: ZO-1, ZO-2 and claudin-5 are

recruited under N. meningitidis colonies (lower Panel). Arrow indicates a bacterial

colony. Scale bars: 10µm. (B) YFP-tagged Par6 (par6-YFP) or myc-tagged Par3

(par3-myc), both green, are recruited underneath N. meningitidis colonies where they

co-localize with actin (red). Areas outlined in white indicate the presence of a N.

meningitidis colony. Scale bars: 10µm. The formation of these ectopic early junction-like

domains is not found underneath all N. meningitidis colonies. Signaling underneath

bacterial microcolonies required a minimal number of 20 bacteria per colony to be

detected by immunofluorescence, with around 40-50% of microcolonies containing 40-50

bacteria. The average number of colonies signalling after 2 hours of infection is 40 %.

Figure 2

The Cdc42-Par3/Par6/PKCζ pathway controls the formation of ectopic early

junction-like domains. (A) Knockdown of Cdc42 was performed using specific siRNA

duplexes (Cdc42 siRNA). Cells were cotransfected with par6-YFP or par3-myc.

Knockdown of Cdc42 by RNAi reduced the recruitment of par6-YFP and par3-myc by 4

fold. * t test (p<0.005). (B) Knockdown of Cdc42, Par6 and Par3 were performed as

siCONTROL were used as control for Cdc42/Par6 and Par3 knockdown, respectively.

Knockdown of Cdc42 by RNAi reduced the recruitment of VE-cadherin, p120-catenin

and actin by 2.2 fold, 2.3 fold and 2.5 fold, respectively. See also figure S3. Knockdown

of Par6 by RNAi reduced the recruitment of VE-cadherin, p120-catenin and actin by 2.7

fold, 2.4 and 2.4 fold, respectively. Knockdown of Par3 by RNAi reduced the recruitment

of VE-cadherin by 2 fold. * t test (p<0.01), ** t test (p<0.002). (C, D) HCMEC/D3 cells

were either incubated with 3µM or 6µM of PKCζ pseudosubstrate inhibitor (PKCζ-PS) or PKCη-PS (control), or left untreated. (C). PKCζ-PS (6µM) reduced VE-cadherin,

p120-catenin and actin recruitment by 8.5 fold, 5 fold and 4.9 fold, respectively. * t test

(p<0.001). (D) HCMEC/D3 cells were transfected with either par6-YFP or par3-myc. Six µM PKCζ-PS reduced par3-myc recruitment by 9 fold, but par6-YFP recruitment was not

affected (* t test (p<0.001), ** t test (p<0.01)). Data are expressed as mean +/- SEM.

Figure 3

P120-catenin is key to the recruitment of both actin and AJ proteins. (A, B)

VE-cadherin silencing was performed by stable expression of a VE-VE-cadherin shRNA (VEC

shRNA). (A) Recruitment of β-catenin, p120-catenin and actin was determined by

immunofluorescence. Knockdown of VE-cadherin had no effect on the recruitment of

p120-catenin and actin but reduced β-catenin recruitment by 20 fold. * t test (p<0.001).

(B) In VEC-shRNA expressing cells, p120-catenin was still recruited beneath N.

meningitidis colonies where it colocalized with actin (upper panel) while β-catenin was

no longer recruited (lower panel). Areas outlined in white indicated the location of a N.

using a specific siRNA duplex (p120 siRNA). Recruitment of VE-cadherin and actin was

determined by immunofluorescence. Knockdown of p120-catenin reduced VE-cadherin

and actin recruitment by 10 fold and 4 fold, respectively. * t test (p<0.001). Data are

expressed as mean +/- SEM.

Figure 4

N. meningitidis induced PKCζ activity facilitates cell-cell junction opening. (A) The

permeability coefficient of Lucifer Yellow was measured 4h post-infection by N.

meningitidis (Nm) or its non piliated isogenic strain (Nm ΔpilE), or following treatment

by PKCζ-PS or PKCη-PS (6µM). N. meningitidis induced a 1.55 fold increase compare to

control. D-mannitol, which disrupts all cell-cell junctions, induced a 3.1 fold increase. * t

test (p<0.001). (B) HCMEC/D3 cells were incubated with 6µM of PKCζ-PS or of

PKCη-PS (control). (a) VE-cadherin localization was analyzed on the baso-lateral cross-section

of N. meningitidis infected cells. Yellow arrow heads and areas outlined in yellow

indicate gaps between cells. Areas outlined in red indicate the presence of N. meningitidis

colonies. Blue bars marked 1-4 refer to Z-axis reconstruction image 1-4 on the lower

panel. Scale bars: 20µm. (b) Z-axis reconstructions from stack of 0.12µm interval images

show that VE-cadherin is apically relocalized underneath N. meningitidis colonies (white

arrows) only in cells treated with PKCη-PS (control). (C) HCMEC/D3 cells grown on

3.0µm pore size inserts were treated or not with PKCζ-PS and incubated with N.

meningitidis (Nm) or its non piliated isogenic strain (N.meningitidis ΔpilE). Size and

quantity of gaps observed 4h after infection are calculated as described (27). (D)

3.2 fold higher than diffusion of N. meningitidis in presence of 6µM PKCζ-PS and 16.5

fold higher than diffusion of its non-piliated derivative (Nm ΔpilE). The rate of N.

meningitidis internalization, determined by gentamicin protection assay, is very low

(1CFU in 3,5.105), identical to that of a control without cells, thus excluding a possible

transcytosis of bacteria. Data are expressed as fold increase of N. meningitidis diffusion

and calculated as described (27). Data from B, C and D are one representative experiment